Phosphoenolpyruvate (PEP) carboxylase serves a number of metabolic functions in bacteria and plants, including a central role in the C4 photosynthetic pathway. While deduced amino acid sequences of the enzyme from several sources are available, little is known about the location or composition of the allosteric sites of this enzyme. The broad goal of this project is to utilize affinity labeling and site-directed mutagenesis to advance our understanding of effector sites and regulatory mechanisms of this enzyme. The specific aims of the project include the following: 1) Compete the remaining work in our current project to isolate and sequence the peptide from the phosphorylated activator binding site which has been labeled with periodate-oxidized AMP (oAMP) 2) Probe the location and specificity of the ATP inhibition site with oAMP and the potential affinity label oATP 3) Compare the physical and kinetic properties of recombinant maize PEP carboxylase from two full length cDNA clones, pGLW5 and pGLW6, and improve the level of expression of these two PEP carboxylase clones in E. coli 4) Use site-directed mutagenesis to probe the function of residues that are suspected of having a regulatory role. The target residues include threonine 227 and several residues in the region near this residue, which is conserved among higher plant and bacterial PEP carboxylases and which is known to be crucial for activation of the enzyme by fructose 1 ,6 bisphosphate. Other target residues are those in the peptide being identified by affinity labeling with oAMP, and ultimately those identified by labeling with oATP. The information from these studies will provide insight into the adenylate and sugar phosphate regulatory sites of this enzyme and, more generally, into the process of metabolic regulation at the enzyme level.